Lipase is a very important metabolic enzyme for ordinary biological body; it can hydrolyze fat to produce free fatty acid. Lipases or acylglycerol hydrolases are enzymes that catalyze the hydrolysis of long chain triglyceride into diacylglyceride, monoglyceride, glycerol and free fatty acids. However, lipases are also capable of catalyzing the reverse reaction of hydrolysis in the formation of esters from alcohols and fatty acids or via transesterification.
The lipase is extensively used as an enzyme for food processing to flavor dairy products, medicines as digestive, improvements of fats and oils, and the like. The lipase is required to have various characteristics for each use, and a thermostable lipase is applied to a wide various characteristics for each use, and a thermostable lipase is applied to a wide variety of fields and requested to be variously used.
Microbial extracellular lipases are usually more thermostable than animals or plants lipases. Microbial extracellular lipase has a potential use in industries and diagnostics. A major requirement for commercial enzyme is thermal stability because thermal denaturation is a common cause of enzyme inactivation. In addition, increasing enzyme thermostability would allow enzymatic reactions to be carried out at higher temperatures; this would help to increase conversion rates, substrate solubility and to reduce the possibility of microbial growth and the viscosity of the reaction medium.
Although thermophiles could be a good candidate in producing thermostable enzyme, but it is often impractical because of low yield and also high temperature fermentation equipment may be needed. To overcome this problem, a molecular approach through genetic engineering becomes a good alternative to achieve high-level expression towards bulk production economically via prokaryotic system. So far, several thermostable lipases have been successfully cloned and expressed in heterologous hosts intracellularly.
For fundamental studies and for commercial purposes expression of foreign protein in prokaryotic systems is most widely used to achieve high-level expression. Expression vector and host are an important issue for achieving maximal expression of cloned gene, however molecular cloning of foreign gene does not ensure that the gene been expressed successfully.
At this present invention, rapid cloning of thermostable lipase through Polymerase Chain Reaction (PCR) technique and manipulation of the thermostable lipase T1 gene is express through prokaryotic system using various kinds of promoters.